Chimeric pestivirus with insertion in 3′ nontranslated region (3′NTR) with stable replication and rnase resistance

Abstract

The construction of a chimeric Pestivirus by the identification of selected regions in the 3′NTR of the viral RNA genome is described where additional RNA sequences can be stably inserted. These sequence insertions in the viral RNA genome were stable in replication and capable of forming infectious, RNase resistant virus particles. This chimeric Pestivirus with a 3′NTR insertion can be utilized as a quality control material in analytical assays for RNA targets, including external, internal controls, quantitative standards in PCR and NAT nucleic acid assays.

What is claimed is: 
     
       1. An in vitro method to replicate chimeric Pestivirus in a host cell comprising:
 inserting into a Pestivirus RNA sequence a second RNA sequence within a variable region of a 3′ non-translated region (NTR) of the Pestivirus RNA sequence downstream of a 5′ UGA box to produce a chimeric Pestivirus RNA sequence, wherein the Pestivirus is bovine viral diarrhea virus (BVDV) and wherein the second RNA sequence is selected from the group consisting of Hepatitis C Virus (HCV), HCV genotypes 1-7, and combinations thereof; 
 transfecting the chimeric Pestivirus RNA sequence into a host cell; and 
 stably replicating the chimeric Pestivirus in the host cell. 
 
     
     
       2. The method of  claim 1  wherein the BVDV is non-CP7 clone. 
     
     
       3. The method of  claim 1  wherein the second RNA sequence is located between the 5′ UGA box and a 3′ UGA-like box. 
     
     
       4. The method of  claim 1  wherein the second RNA sequence is inserted between the SL STOP  stem-loop and an SLII stem-loop. 
     
     
       5. The method of  claim 1  wherein the second RNA sequence is located between a UGA pos.cons  box and the SLII stem-loop. 
     
     
       6. The method of  claim 1  wherein the second RNA sequence is a portion of a Hepatitis C virus (HCV). 
     
     
       7. The method of  claim 6  wherein the portion of the HCV virus is comprised of at least 8 nucleotides. 
     
     
       8. The method of  claim 6  wherein the second RNA sequence is at least a portion of a 5′ NTR region of the HCV virus.